Guifang Cheng

An aptamer-based assay for thrombin via structure switch based on gold nanoparticles and magnetic nanoparticles.

An aptamer-based assay for thrombin with high specificity and sensitivity was presented. In the protocol, the aptamer for thrombin was immobilized on magnetic nanoparticle, and its complementary oligonucleotide was labeled with gold nanoparticles, then the aptamer was hybridized with the complementary oligonucleotide to form the duplex structure as a probe, this probe could be used for the specific recognition for thrombin. In the presence of thrombin, the aptamer prefer to form the G-quarter structure with thrombin, resulting in the dissociation of the duplex of the probe and the release of the gold labeled oligonucleotide. Upon this, we were able to detect thrombin through the detection of the electrochemical signal of gold nanoparticles. The strategy combines with the high specificity of aptamer and the excellent characteristics of nanoparticles. This assay is simple, rapid, sensitive and highly specific, it does not require labeling of thrombin, and it could be applied to detect thrombin in complex real sample. The method shows great potential in other protein analysis and in disease diagnosis.

Simultaneously fluorescence detecting thrombin and lysozyme based on magnetic nanoparticle condensation

In this protocol, a fluorescent aptasensor based on magnetic separation for simultaneous detection thrombin and lysozyme was proposed. Firstly, one of the anti-thrombin aptamer and the anti-lysozyme aptamer were individually immobilized onto magnetic nanoparticles, acting as the protein captor. The other anti-thrombin aptamer was labeled with rhodamine B and the anti-lysozyme aptamer was labeled with fluorescein, employing as the protein report. By applying the sandwich detection strategy, the fluorescence response at 515 nm and 578 nm were respectively corresponding to lysozyme and thrombin with high selectivity and sensitivities. The fluorescence intensity was individually linear with the concentration of thrombin and lysozyme in the range of 0.13-4 nM and 0.56-12.3 nM, and the detection limits were 0.06 nM of thrombin and 0.2 nM of lysozyme, respectively. The preliminary study on simultaneous detection of thrombin and lysozyme in real plasma samples was also performed. It shows that the proposed approach has the good character for simultaneous multiple protein detection.

A new electrochemically active–inactive switching aptamer molecular beacon to detect thrombin directly in solution

A new electrochemical aptamer molecular beacon (MB) was designed by the carminic acid (CA) covalently linking at the each end of a special single-stranded stem-loop shaped oligonucleotide and named as CAs-MB. CA is an electrochemically active molecule and two CA molecules at the ends of molecular beacon stem were closed enough to associate each other to be as CA dimer. The dimer was electrochemically inactive. It separated into two CA monomers and produced the electrochemical signal while CAs-MB combined with target. In this protocol, the detection strategy of CAs-MB for thrombin is based on electrochemical active-inactive switching between monomer and dimer forms of CA. In order to enhance the electrochemical signal, magnetic nanobeads (MNB) was applied by connecting CAs-MB with MNB through a duplex of DNA. With the magnetic enrichment, the detection limit for thrombin reached to 42.4 pM. The experiment results showed that this type of electrochemical active-inactive switching aptamer molecular beacon allowed the direct detection of target proteins in the solution with no requirement of removing uncombined CAs-MB. Besides, CAs-MB/MNB can be easily regenerated by using 2M NaCl solution to cleave the thrombin from the aptasensor.

A novel optical thrombin aptasensor based on magnetic nanoparticles and split DNAzyme

In this paper, we report a novel and sensitive optical sensing protocol for thrombin detection based on magnetic nanoparticles (MNPs) and thrombin aptamer, employing split HRP-mimicking DNAzyme halves as its sensing element, which can catalyze the H(2)O(2)-mediated oxidation of the colorless ABTS into a blue-green product. A single nucleotide containing the recognition element and sensing element is utilized in our protocol. The specific recognition of thrombin and its aptamer leads to the structure deformation of the DNA strands and causes the split of the DNAzyme halves. Therefore, the decrease of absorption spectra can be recorded by the UV-visible Spectrophotometer. DNA-coated MNPs are utilized to separate the interferential materials from the analyst, thus making this assay can be applied in the detection of thrombin in complex samples, such as human plasma. This original, sensitive and cost-effective assay showed favorable recognition for thrombin. The absorbance signals with the concentration of thrombin over a range from 0.5 to 20 nM and the detection limit of thrombin was 0.5 nM. The controlled experiments showed that thrombin signal was not interfered in the presence of other co-existence proteins.

A Novel Electrochemical Biosensor for Deoxyribonucleic Acid Detection Based on Magnetite Nanoparticles

Abstract DNA detection is of great importance for clinical diagnosis and food safety. In the present study, the authors proposed a sensitive electrochemical biosensor for DNA sequence mutation detection based on the separation and enrichment of magnetic nanobeads. For the proposed biosensor, the probe sequences were immobilized on the surface of the carboxyl functioned magnetic nanobead, to recognize the target sequences, and methylene blue was used as a hybridization indicator. In the range of 1.0 × 10−13–1.0 × 10−6M, the concentration of the complementary sequence was linear with the response of the electrochemical signal of MB and the detective limit of target oligonucleotide was 43 fM. The improvement in the detective sensitivity of this biosensor was mainly due to magnetic enrichment and the multiwall carbon nanotube modified electrode.

Spectroelectrochemical study of the interaction between antitumor drug daunomycin and DNA in the presence of antioxidants

Anthracycline drug daunomycin (DNR) is a widely used clinical drug. But its side effects, especially cardiotoxicity, have greatly restrained its application. The side effects were due to free radical formation in the metabolic process of DNR. The purpose of this study is to diminish the side effects by using antioxidants. Two kinds of free radical scavengers have been investigated, that is, vitamins: vitamin C (V(C)), rutin (V(P)) and vitamin B6 (V(B6)); amino acids: cysteine (CysH) and methionine (Met). Free radical scavenging efficiency (E degrees (eff)) of these antioxidants had been calculated. Under the experimental condition, the values of E degrees (eff) of V(C), V(P), V(B6), CysH and Met were 23.8, 15.3, 6.4, 48.2 and -7.7%, respectively. The relationship between the free radical scavenging activities and its chemical structure has also been discussed.

An ultra-sensitive colorimetric Hg2+-sensing assay based on DNAzyme-modified Au NP aggregation, MNPs and an endonuclease

This paper reports the development of an ultra-sensitive colorimetric method for the detection of trace mercury ions involving DNAzymes, Au nanoparticle aggregation, magnetic nanoparticles and an endonuclease. DNAzyme-sensing elements are conjugated to the surface of Au nanoparticle-2, which can crosslink with the T-rich strands coated on Au nanoparticle-1 to form Au nanoparticle aggregation. Other T-rich stands are immobilized on the surface of MNPs. The specific hybridization of these two T-rich strands depends on the presence of Hg(2+), resulting in the formation of a T-Hg(2+)-T structure. Added endonuclease then digests the hybridized strands, and DNAzyme-modified Au NP aggregation is released, catalysing the conversion of the colourless ABTS into a blue-green product by H2O2-mediated oxidation. The increase in the adsorption spectrum of ABTS(+) at 421 nm is related to the concentration of Hg(2+). This assay was validated by detecting mercury ion concentrations in river water. The colorimetric responses were not significantly altered in the presence of 100-fold excesses of other metal ions such as Zn(2+), Pb(2+), Cd(2+), Mn(2+), Ca(2+) and Ni(2+). The inclusion of both Au NP aggregation and an endonuclease enables the assay to eliminate interference from the magnetic nanoparticles with colorimetric detection, decrease the background and improve the detection sensitivity. The calibration curve of the assay was linear over the range of Hg(2+) concentrations from 1 to 30 nM, and the detection limit was 0.8 nM, which is far lower than the 10 nM US EPA limit for drinking water.

Au Nanoparticle-DNAzyme Dual Catalyst System for Sensitively Colorimetric Detection of Thrombin

A novel colorimetric aptasensor was developed for thrombin detection with high sensitivity and specificity. The assay takes the advantage of Au nanoparticles-DNAzyme as a dual catalytic system for signal amplification. Au nanparticles were modified with peroxidase mimicking DNAzyme sequence as well as thrombin binding aptamer. And then the thrombin binding aptamer hybridized with its complementary sequence which was immobilized on the surface of the magnetic nanoparticles to construct the colorimetric aptasensor. In the presence of thrombin, the target-induced displacement takes place, resulting in the dissociation of the aptasensor. The DNAzyme functioned Au NPs are released due to the combine ability of thrombin binding aptamer with thrombin. The released Au NPs are capable of catalyzing the colorless 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid(ABTS) conversion into a blue-green product by H2O2-mediated oxidation, thus can amplify the colorimetric readout signals of thrombin detection. Such a device can serve as a novel selectivity sensor for thrombin with a detection limit of 0.6 nmol/L.

Design strategy for a novel electrochemically active–inactive switching molecular beacon based on Hemin for SNPs and insulin detection directly in homogenous solution

Herein, a novel and convenient electrochemically active-inactive switching molecular beacon based on hemin (Hs-MB) has been designed for easy discrimination of single nucleotide polymorphisms (SNPs) and sensitive detection of insulin. The electrochemically active changing capability of Hs-MB is based on two identical hemin groups labeled at both ends of MB sequence in dimer or monomer forms depending on the conformation of MB which is in stem-loop structure or line shaped structure. The Hs-MB assay permits discrimination of SNPs and the highly sensitive and specific detection of insulin with detection limit successively as low as 0.5 pmol/L. Even at a very low target concentration, the Hs-MB assay also shows a good specificity in the presence of other potentially interfering components. The experimental results also show that Hs-MB can also be used for the accurate and rapid monitoring of insulin secretion by glucose-stimulated from MIN6 cells at different time periods, demonstrating that Hs-MB has potential in monitoring of biomarker variation in vivo.

A sensitive colorimetric biosensor based on intermolecular split G-quadruplex DNAzymes and magnetic nanoparticles for the detection of Hg2+

In this paper, we report a novel and sensitive optical sensing protocol for the detection of mercury (II) ions based on colorimetry combined with intermolecular split G-quadruplex DNAzymes and the conjugation of thymine-Hg2+-thymine. A T-rich strand A was assembled onto the biomagnetic beads. Two other oligonucleotide strands (strands B and C) were employed, which are each composed of a capture segment (T-rich part) and a sensing segment (G-rich part). In the presence of Hg2+, the capture segments of both strands B and C can hybridise with biotin-strand A on magnetic beads to form the stable DNA duplexes through T-Hg2+-T linkages. The formation of the DNA duplex brings the sensing segments of both strands B strand C close enough to constructed an intermolecular split G–quadruplex. This split G-quadruplex is able to effectively catalyse the H202-mediated oxidation of ABTS in the presence of hemin, generating the blue-green product ABTS+ and increasing the absorption signal at 421 nm. Therefore, the change in the absorption signal at 421 nm can be used to monitor the concentration of Hg2+. The calibration curve of the assay was linear over the range of Hg2+ concentrations from 2 to 50 nM, and the detection limit was 0.8 nM. Moreover, the influence of the intermolecular split G-quadruplex DNAzyme strands on the catalytic ability is also discussed. The practical results obtained imply that the proposed colorimetric biosensor can be extended for the detection of trace Hg2+ in environmental water and drinking water.